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. 2019 Jul 31;9(1):11137.
doi: 10.1038/s41598-019-47319-w.

A Fiber-Coupled Stimulated Emission Depletion Microscope for Bend-Insensitive Through-Fiber Imaging

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Free PMC article

A Fiber-Coupled Stimulated Emission Depletion Microscope for Bend-Insensitive Through-Fiber Imaging

Brendan M Heffernan et al. Sci Rep. .
Free PMC article

Abstract

We present results for a new type of fiber-coupled stimulated emission depletion (STED) microscope which uses a single fiber to transport STED and excitation light, as well as collect the fluorescence signal. Our method utilizes two higher-order eigenmodes of polarization maintaining (PM) fiber to generate the doughnut-shaped STED beam. The modes are excited with separate beams that share no temporal coherence, yielding output that is independent of fiber bending. We measured the resolution using 45 nm fluorescent beads and found a median bead image size of 116 nm. This resolution does not change as function of fiber bending radius, demonstrating robust operation. We report, for the first time, STED images of fixed biological samples collected in the epi-direction through fiber. Our microscope design shows promise for future use in super-resolution micro-endoscopes and in vivo neural imaging in awake and freely-behaving animals.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Simulated addition of PM fiber modes. (a) and (b) When the two excited fiber modes are coherent with respect to each other, i.e. their phases are correlated, the resulting mode depends on their relative phase. This means that fiber bending will change the output by changing the relative phase. (c) If the two modes share no phase correlation, the resulting mode is a doughnut shape regardless of relative phase.
Figure 2
Figure 2
A diagram of the fiber STED microscope. Fluorophores are excited at 485 nm and depleted at 585 nm. The lasers have a repetition rate of 20 MHz. The STED laser (at 585 nm) is shaped to match the PM01 mode using a spatial light modulator (SLM). A polarizing cube beamsplitter (PBS) splits the beam into a Mach-Zehnder interferometer. In one arm, a dove prism rotates the spatial profile of the laser and introduces a delay of 74 ps. The transverse modes of the two STED beams are pictured, with the polarization indicated by black arrows. These beams are combined using another PBS and coupled to PM fiber (two meter length). The excitation light is sent through quarter and half-wave plates to control its polarization and then a high-pass dichroic mirror combines it with the STED light. Upon exiting the fiber, the STED and excitation beams are collimated and focused onto a fluorescent sample using a 1.4 NA, oil-immersion objective. This objective collects and collimates fluorescence, which is then coupled back into the PM fiber. A dichroic mirror splits the fluorescence from the common beam path. The light is passed through two chromatic filters and then focused into a multimode (MM) fiber coupled to a single photon counting module (SPCM).
Figure 3
Figure 3
Images of 45 nm fluorescent beads attained using (a) confocal and (b) STED modality. There was approximately 45 mW of power in the STED laser before the objective. The pixels size is 19.5 nm. These images have been convolved with a small Gaussian (waist of 0.8 pixels) for smoothing and the background has been subtracted to enhance clarity. A magnified area of interest is shown, and (c) a cross sectional cut of the boxed region within the magnification is shown. The top right corner of the magnified area in (a) and (b) shows raw confocal and STED images from the microscope for comparison. (d) Histogram of Gaussian-fitted FWHM values for bead images. The confocal distribution has a median of 260 nm, while the STED distribution has a median of 116 nm. This demonstrates more than a two-fold improvement of resolution from confocal. Both (c) and (d) were derived from raw data.
Figure 4
Figure 4
Images of HeLa cells immunostained for tubulin using Alexa 488. The images have been convolved with a Gaussian with a waist of 0.8 pixels (39 nm) for smoothing and the background was subtracted. The top row are confocal and the bottom row are STED images. Normalized linecuts of raw data are shown in (a) and (b), demonstrating at least a two-fold improvement in resolution. The pixel size is 48.8 nm and approximately 20 mW of STED and 6 μW of excitation power were used, measured before the objective. Figure reproduced from.
Figure 5
Figure 5
Box plot of the measured FWHM of bead images for 100 nm fluorescent beads. The center line in each box gives the median of the data set and the outer edges of the box represent the 25th and 75th percentile. The dashed line and bar denote the extremes of the distribution, not considering statistical outliers. Note that while the distribution of FWHM’s changes, this is caused by a time drift in the z-axis of the stage rather than by changes in fiber conditions. Below are images of the fiber in bent states, with minimum bend radius marked. Figure reproduced from.

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